This application is related to Merck Case 19262, U.S. Ser. No. 08/323,953, filed Oct. 17, 1994.
The human cytomegalovirus (HCMV) is the etiological agent of a variety of infectious diseases in infants and children. The virus also is involved in severe infections of adults with immunodeficiencies, such as AIDS patients or transplant recipients. The HCMV encodes a protease that participates in the maturation of the viral capsid. The enzyme processes the viral assembly protein within the capsid core by mediating cleavage between the ala-ser peptide bond at residue positions 308/309. This results in the linked extrusion of the assembly protein and the encapsidation of the viral genomic DNA.
The association of the individual assembly proteins into the capsid likely results from specific intermolecular protein interactions. The presence of the protease at the N-terminus of a 80 kD precursor that also contains the assembly protein assures localization of the enzyme in the capsid as a consequence of interactions mediated by the assembly protein portion. A mutant of the herpes simplex virus type 1 (HSV-1), which expresses temperature-sensitive alterations in the protease, is incapable of processing the assembly protein and encapsidating genomic DNA at the non-permissive temperature. This result indicates that a specific potent inhibitor of the viral enzyme would be useful as a therapeutic agent.
Applicants have discovered that a highly active form of HCMV protease is a dimer of identical units. At ambient or higher temperatures, most of the protease dissociates into its monomeric form which is inactive as gauged by kinetic assays. For this reason, the activity of this protease has been erroneously believed to be very low. Having an equilibrium of monomer and dimer of an enzyme in solution with the inactive monomer at high concentration will obscure detection of inhibitors of the active, dimeric form of the enzyme. This physiochemical phenomenon is governed by thermodynamic principles. Due to the high activation energy required for dissociation, the HCMV protease can be "frozen" in its active, dimeric form at about 0.degree. C., thus enabling sensitive detection of inhibitors of this enzyme. The assay of this present invention provides a screening protocol, conveniently carried out at 0.degree. C., for inhibitors of the HCMV protease. The existence of the HCMV protease has been reported, e.g., see Pinko, et al., J. Biol. Chem. 270, 23634 (1995). Assays for the catalytic activity of this enzyme have been described, albeit the reported kinetic parameters were substantially lower than those measured by applicants. Since the dissociation of the dimeric HCMV protease into its inactive monomeric form is responsible for the apparent low activity of the enzyme, the enzyme is "frozen" in its dimeric form by lowering the temperature of the assay and related operations to about 0.degree. C. This approach affords an assay for the activity of the protease at low concentration (.ltoreq.20 nM), thus permitting detection of weak inhibitors or strong inhibitors at low concentration.
The resulting highly active assay for HCMV protease is useful as a screening tool for HCMV antivirals in the potency range of about 100-200 nM (or less), as well as a diagnostic tool for diseases resulting from HCMV infection.